Human cytomegalovirus (HCMV) is a significant opportunistic pathogen causing severe disease in newborns infected in utero and the immunocompromised. HCMV replication requires a cascade of gene expression, consisting of immediate early (IE), early and late phases. As products of the early genes play key roles in both pathogenesis and DNA replication, transition into this phase is critical in determining the outcome of viral infection. The objective of this application is to determine how viral and cellular proteins control transcription of viral genes during this transition. Our hypothesis is that two viral proteins, pp71 and IE86, play key roles in this process via direct interaction and/or alteration of the nuclear environment. This hypothesis will be tested in four aims: (1) Characterize the role of cellular factors in US6 regulation in the viral genome. We will determine the role of ATF and Spl in the activation of the US6 early gene in the natural genomic environment. (2) Determine the role of IE86 interactions with cellular proteins in the activation of viral early promoters. We hypothesize that co-activator functions of IE86 play a critical role in the activation of viral early genes. To assess this, mutants within a key region from amino acids 535 to 545 will be used to define the properties of IE86 that are essential for transcriptional regulation of viral early promoters. (3) Determine the mechanism of transactivation by the viral tegument protein pp71. Our hypothesis is that pp71 regulates viral gene expression via direct interaction with cellular transcription factors. This hypothesis will be tested by identifying domains of pp71 required for both protein-protein interactions and transcriptional regulation. (4) Determine the mechanism of transcriptional cooperation between IE86 and pp71. We hypothesize that direct and/or functional interaction between IE86 and pp71 is important in regulating viral gene expression during the transition from the IE to early phase. This hypothesis will be tested by determining the domains required for functional association between these two proteins and assessing the effects on transcriptional activation. The studies that we describe in this proposal will therefore enhance our understanding of the mechanisms of transcriptional activation of viral early genes and may lead to the development of novel antiviral therapies.